Prostaglandin receptor EP2

ABSTRACT

A novel prostaglandin receptor has been identified and DNA encoding the receptor has been isolated, purified, sequenced and expressed in host cells. This DNA encoding the novel prostaglandin receptor and host cells expressing the receptor are used to identify modulators of the prostaglandin receptor.

This application is the U.S. national phase (35 U.S.C. §371) ofInternational Patent Application No. PCT/CA94/00470, filed Aug. 29,1994, which is a continuation-in-part of U.S. patent application Ser.No. 08/115,365, filed Aug. 31, 1993, now U.S. Pat. No. 5,605,814.

BACKGROUND OF THE INVENTION

The physiological actions of prostaglandin (PG)E₂ are mediated throughinteraction with the prostaglandin E receptor(s). There are threesubtypes of the EP receptor, EP₁, EP2 and EP₃ (for review see Coleman etal., 1989). These three subtypes all show high affinity for PGE2 butshow differences in their affinities for various agonists andantagonists and exert their actions through different secondarytransduction mechanisms. Thus activation of the EP1 receptor isassociated with a rise in IP3 and intracellular calcium, activation ofthe EP2 receptor results in a rise in intracellular cyclic AMP andactivation of the EP3 receptor a fall in intracellular cyclic AMP. Todate the only members of this family to be cloned are the mouse EP2(Honda et al., 1993) and the mouse EP₃α and EP₃β (Sugimoto et al., 1992;Sugimoto et al., 1993) subtypes. EP2 receptors are normally found on awide variety of cells including the small intestine, kidney, stomach,muscle, eye, uterus, thymus and trachea, in humans and other animals.Binding of prostaglandin E₂ to the EP2 receptor protein elicits anincrease in intracellular cAMP levels. This signal causes the tissues torespond, for example, by smooth muscle relaxation.

Functional activities of the EP2 receptor have been studied using tissuepreparations such as guinea-pig ileum circular muscle, cat trachea,guinea-pig trachea and cell preparations, such as lymphocytes andosteoclasts. The above methods for studying EP2 receptor activities haveseveral disadvantages in that they require preparations containingseveral different but related receptor populations, with differentligand binding properties making measurements of absolute potency andselectivity very difficult. In addition, tissues contain very low levelsof EP2 receptor and since tissue samples are required, compounds cannotsatisfactorily be tested as effectors of the human EP2 receptor.

SUMMARY OF THE INVENTION

A novel prostaglandin receptor protein termed EP2 has been isolated andpurified from human cells. A DNA molecule encoding the full length EP2protein has been isolated and purified, and the nucleotide sequence hasbeen determined. The EP2 encoding DNA has been cloned into expressionvectors and these expression vectors, when introduced into recombinanthost cells, cause the recombinant host cells to express a functional EP2receptor protein. The novel EP2 protein, the EP2-encoding DNA, theexpression vectors and recombinant host cells expressing recombinant EP2are useful in the identification of modulators of EP2 receptor activity.

A method of identifying EP2 receptor modulators is also disclosed whichutilizes the recombinant EP2 expressing host cells. Modulators of EP2activity are useful for the treatment of prostaglandin-related diseasesand for modulating the effects of prostaglandins on the EP2 receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C--The complete DNA sequence encoding the EP2 receptor proteinis shown above the complete deduced amino acid sequence of the EP2receptor protein.

FIG. 2--Expression of the prostaglandin E2 receptor in EP2 cDNA-injectedXenopus oocytes is shown by an inward cAMP-dependent Cl⁻ current (shownas downward deflection) evoked by bath perfusion of 1 nM PGE2 when theoocyte was injected with 1.6 ng EP2 cDNA plus 2.5 ng CFTR cDNA andvoltage-clamped at -60 mV.

FIG. 3--IBMX-induced, CFTR-mediated Cl⁻ current in (CFTR+anti-sensehEP2) cDNA injected oocytes is shown, noting the lack of response to 1μM and 3 μM PGE2.

FIGS. 4A and B--Competition for ³ H!PGE2 specific binding topcDNAIamp-hEP2 transfected COS-M6 membranes is shown by ³ H!PGE2 bindingassays performed in the presence of: Panel A) 10 pM-10 mM PGE2 (l), PGE1(o), 17-pheyyl-trinor PGE2 (n), iloprost (q), PGF2α (u), PGD₂ (⋄), andU46619 (s) and Panel B, 100 pM-100 uM MB28767 (l), misoprostol (o),butaprost (n), AH6809 (q) and SC19220 (u).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to cDNA encoding a novel prostaglandinreceptor, termed EP2. The present invention is also related torecombinant host cells which express the cloned EP2-encoding DNAcontained in a recombinant expression plasmid. The present invention isalso related to methods for the screening of substances which modulateEP2 receptor activity. The DNA of the present invention is isolated fromEP2 producing cells. EP2, as used herein, refers to a G protein-coupledreceptor which can specifically bind prostaglandin molecules.

Mammalian cells capable of producing EP2 include, but are not limitedto, cells derived from small intestine, kidney, stomach, vascular smoothmuscle, eye, placenta, uterus, lyphocytes, osteoclasts and thetracheobronchial tree. Transformed mammalian cell lines which produceEP2 include, but are not limited to, mastocytoma P-815 cells. Thepreferred cells for the present invention include normal human kidneyand lung cells and the most preferred cells are human thymus cells.

Other cells and cell lines may also be suitable for use to isolate EP2cDNA. Selection of suitable cells may be done by screening for EP2 oncell surfaces. Methods for detecting EP2 activity are well known in theart and measure the binding of radiolabelled ligand specific for thereceptor. Cells which possess EP2 activity in this assay may be suitablefor the isolation of EP2 cDNA.

Any of a variety of procedures may be used to clone EP2 cDNA. Thesemethods include, but are not limited to, direct functional expression ofthe EP2 cDNA following the construction of an EP2-containing cDNAlibrary in an appropriate expression vector system. Another method is toscreen an EP2-containing cDNA library constructed in a bacteriophage orplasmid shuttle vector with a labelled oligonucleotide probe designedfrom the amino acid sequence of the EP2 protein. The preferred methodconsists of screening an EP2-containing cDNA library constructed in abacteriophage or plasmid shuttle vector with a partial cDNA encoding theEP2 protein. This partial cDNA is obtained by the specific PCRamplification of EP2 DNA fragments through the design of degenerateoligonucleotide primers from the amino acid sequence known for other Gprotein-coupled receptors which are related to the prostaglandin EP2receptors.

It is readily apparent that other types of libraries, as well aslibraries constructed from other cells or cell types, may be useful forisolating EP2-encoding DNA. Other types of libraries include, but arenot limited to, cDNA libraries derived from other cells or cell linesand genomic DNA libraries.

It is readily apparent that suitable cDNA libraries may be prepared fromcells or cell lines which have EP2 activity. The selection of cells orcell lines for use in preparing a cDNA library to isolate EP2 cDNA maybe done by first measuring cell associated EP2 activity using the knownlabelled ligand binding assay cited above and used herein.

Preparation of cDNA libraries can be performed by standard techniqueswell known in the art. Well known cDNA library construction techniquescan be found for example, in Maniatis, T., Fritsch, E. F., Sambrook, J.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,Cold Spring Harbor, New York, 1982).

It is also readily apparent that DNA encoding EP2 may also be isolatedfrom a suitable genomic DNA library. Construction of genomic DNAlibraries can be performed by standard techniques well known in the art.Well known genomic DNA library construction techniques can be found inManiatis, T., Fritsch, E. F., Sambrook, J. in Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,New York, 1982).

In order to clone the EP2 gene by one of the preferred methods, theamino acid sequence or DNA sequence of EP2 or a homologous protein isnecessary. To accomplish this, EP2 protein or a homologous protein maybe purified and partial amino acid sequence determined by automatedsequenators. It is not necessary to determine the entire amino acidsequence, but the linear sequence of two regions of 6 to 8 amino acidscan be determined for the PCR amplification of a partial EP2 DNAfragment.

Once suitable amino acid sequences have been identified, the DNAsequences capable of encoding them are synthesized. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the EP2 sequence but others in the setwill be capable of hybridizing to EP2 DNA even in the presence of DNAoligonucleotides with mismatches. The mismatched DNA oligonucleotidesmay still sufficiently hybridize to the EP2 DNA to permit identificationand isolation of EP2 encoding DNA.

Using one of the preferred methods, cDNA clones encoding EP2 areisolated in a two-stage approach employing polymerase chain reaction(PCR) based technology and cDNA library screening. In the first stage,NH₂ -terminal and internal amino acid sequence information from thepurified EP2 or a homologous protein is used to design degenerateoligonucleotide primers for the amplification of EP2-specific DNAfragments. In the second stage, these fragments are cloned to serve asprobes for the isolation of full length cDNA from cDNA libraries.

The sequence for the near full-length cDNA encoding EP2 is shown inTable 1, and was designated clone EP2. The deduced amino acid sequenceof EP2 from the cloned cDNA is shown in Table 2. Inspection of thedetermined cDNA sequence reveals the presence of a single, large openreading frame that encodes for a protein of approximately 488 aminoacids.

The cloned EP2 cDNA obtained through the methods described above may berecombinantly expressed by molecular cloning into an expression vectorcontaining a suitable promoter and other appropriate transcriptionregulatory elements, and transferred into prokaryotic or eukaryotic hostcells to produce recombinant EP2. Techniques for such manipulations canbe found described in Maniatis, T, et al., supra, and are well known inthe art.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned DNA and the translation of their mRNAsin an appropriate host. Such vectors can be used to express eukaryoticDNA in a variety of hosts such as bacteria, bluegreen algae, plantcells, insect cells and animal cells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal cells. An appropriatelyconstructed expression vector should contain: an origin of replicationfor autonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one which causes mRNAs to be initiated at highfrequency. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant EP2 in mammalian cells. Commercially available mammalianexpression vectors which may be suitable for recombinant EP2 expression,include but are not limited to, pMClneo (Stratagene), pXT1 (Stratagene),pSG5 (Stratagene), pcDNAI, pcDNAIamp (Invitrogen), EBO-pSV2-neo (ATCC37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),pUCTag (ATCC 37460), 1ZD35 (ATCC 37565), and vaccinia virus transfervector pTM1.

DNA encoding EP2 may also be cloned into an expression vector forexpression in a host cell. Host cells may be prokaryotic or eukaryotic,including but not limited to bacteria, yeast, mammalian cells includingbut not limited to cell lines of human, bovine, porcine, monkey androdent origin, and insect cells including but not limited to drosophiladerived cell lines. Cell lines derived from mammalian species which maybe suitable and which are commercially available, include but are notlimited to, CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658),HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5(ATCC CCL 171).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, infection, protoplast fusion, and electroporation. Theexpression vector-containing cells are individually analyzed todetermine whether they produce EP2 protein. Identification of EP2expressing cells may be done by several means, including but not limitedto immunological reactivity with anti-EP2 antibodies, and the presenceof host cell-associated EP2 activity.

Expression of EP2 DNA may also be performed using in vitro producedsynthetic mRNA. Synthetic mRNA can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being preferred.

To determine the EP2 cDNA sequence(s) that yields optimal levels ofreceptor activity and/or EP2 protein, EP2 cDNA molecules including butnot limited to the following can be constructed: the full-length openreading frame of the EP2 cDNA and various constructs containing portionsof the cDNA encoding only specific domains of the receptor protein orrearranged domains of the protein. All constructs can be designed tocontain none, all or portions of the 5' and/or 3' untranslated region ofEP2 cDNA. EP2 activity and levels of protein expression can bedetermined following the introduction, both singly and in combination,of these constructs into appropriate host cells. Following determinationof the EP2 cDNA cassette yielding optimal expression in transientassays, this EP2 cDNA construct is transferred to a variety ofexpression vectors (including recombinant viruses), including but notlimited to those for mammalian cells, plant cells, insect cells,oocytes, E. coli, and yeast cells.

Mammalian cell transfectants are assayed for both the levels of EP2receptor activity and levels of EP2 protein by the following methods.Assessing EP2 receptor activity involves the direct introduction of alabelled ligand to the cells and determining the amount of specificbinding of the ligand to the EP2-expressing cells. Binding assays forreceptor activity are known in the art (Frey et al., 1993, Eur. J.Pharmacol., 244, pp 239-250).

Levels of EP2 protein in host cells is quantitated by a variety oftechniques including, but not limited to, immunoaffinity and/or ligandaffinity techniques. EP2-specific affinity beads or EP2-specificantibodies are used to isolate ³⁵ S-methionine labelled or unlabelledEP2 protein. Labelled EP2 protein is analyzed by SDS-PAGE. UnlabelledEP2 protein is detected by Western blotting, ELISA or RIA assaysemploying EP2 specific antibodies.

Following expression of EP2 in a host cell, EP2 protein may be recoveredto provide EP2 in active form, capable of binding EP2-specific ligands.Several EP2 purification procedures are available and suitable for use.Recombinant EP2 may be purified from cell membranes by variouscombinations of, or individual application of standard separationtechniques including but not limited to detergent solubilization, saltfractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography.

In addition, recombinant EP2 can be separated from other cellularproteins by use of an immuno-affinity column made with monoclonal orpolyclonal antibodies specific for full length nascent EP2, orpolypeptide fragments of EP2.

Monospecific antibodies to EP2 are purified from mammalian antiseracontaining antibodies reactive against EP2 or are prepared as monoclonalantibodies reactive with EP2 using the technique of Kohler and Milstein,Nature 256: 495-497 (1975). Monospecific antibody as used herein isdefined as a single antibody species or multiple antibody species withhomogenous binding characteristics for EP2. Homogenous binding as usedherein refers to the ability of the antibody species to bind to aspecific antigen or epitope, such as those associated with the EP2, asdescribed above. EP2 specific antibodies are raised by immunizinganimals such as mice, rats, guinea pigs, rabbits, goats, horses and thelike, with an appropriate concentration of EP2 or a peptide derived fromthe sequence of the EP2 protein either with or without an immuneadjuvant.

Preimmune serum is collected prior to the first immunization. Eachanimal receives between about 0.1 μg and about 1000 μg of EP2 orEP2-related peptide associated with an acceptable immune adjuvant. Suchacceptable adjuvants include, but are not limited to, Freund's complete,Freund's incomplete, alum-precipitate, water in oil emulsion containingCorynebacterium parvum and tRNA. The initial immunization consisted ofthe enzyme in, preferably, Freund's complete adjuvant at multiple siteseither subcutaneously (SC), intraperitoneally (IP) or both. Each animalis bled at regular intervals, preferably weekly, to determine antibodytiter. The animals may or may not receive booster injections followingthe initial immunization. Those animals receiving booster injections aregenerally given an equal amount of EP2 or EP2-related peptide inFreund's incomplete adjuvant by the same route. Booster injections aregiven at about three week intervals until maximal titers are obtained.At about 7 days after each booster immunization or about weekly after asingle immunization, the animals are bled, the serum collected, andaliquots are stored at about -20° C.

Monoclonal antibodies (mAb) reactive with EP2 or a peptide derived fromthe sequence of the EP2 protein are prepared by immunizing inbred mice,preferably Balb/c, with EP2 or EP2-related peptide. The mice areimmunized by the IP or SC route with about 1 μg to about 100 μg,preferably about 10 μg, of EP2 or EP2-related peptide in about 0.5 mlbuffer or saline incorporated in an equal volume of an acceptableadjuvant, as discussed above. Freund's complete adjuvant is preferred.The mice receive an initial immunization on day 0 and are rested forabout 3 to about 30 weeks. Immunized mice are given one or more boosterimmunizations of about 1 to about 100 μg of EP2 in a buffer solutionsuch as phosphate buffered saline by the intravenous (IV) route.Lymphocytes, from antibody positive mice, preferably spleniclymphocytes, are obtained by removing spleens from immunized mice bystandard procedures known in the art. Hybridoma cells are produced bymixing the splenic lymphocytes with an appropriate fusion partner,preferably myeloma cells, under conditions which will allow theformation of stable hybridomas. Fusion partners may include, but are notlimited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, withSp 2/0 being preferred. The antibody producing cells and myeloma cellsare fused in polyethylene glycol, about 1000 mol. wt., at concentrationsfrom about 30% to about 50%. Fused hybridoma cells are selected bygrowth in hypoxanthine, thymidine and aminopterin supplementedDulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.Supernatant fluids are collected from growth positive wells on aboutdays 14, 18, and 21 and are screened for antibody production by animmunoassay such as solid phase immunoradioassay (SPIRA) using EP2 orEP2-related peptide as the antigen. The culture fluids are also testedin the Ouchterlony precipitation assay to determine the isotype of themAb. Hybridoma cells from antibody positive wells are cloned by atechnique such as the soft agar technique of MacPherson, Soft AgarTechniques, in Tissue Culture Methods and Applications, Kruse andPaterson, Eds., Academic Press, 1973.

Monoclonal antibodies are produced in vivo by injection of pristineprimed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10⁶ toabout 6×10⁶ hybridoma cells about 4 days after priming. Ascites fluid iscollected at approximately 8-12 days after cell transfer and themonoclonal antibodies are purified by techniques known in the art.

In vitro production of anti-EP2 mAb is carried out by growing thehydridoma in DMEM containing about 2% fetal calf serum to obtainsufficient quantities of the specific mAb. The mAb are purified bytechniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of EP2 inbody fluids or tissue and cell extracts.

It is readily apparent that the above described methods for producingmonospecific antibodies may be utilized to produce antibodies specificfor EP2 polypeptide fragments, or full-length EP2 polypeptide.

EP2 antibody affinity columns are made by adding the antibodies toAffigel-10 (Biorad), a gel support which is pre-activated withN-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) together withappropriate membrane solubilizing such as detergents and the cellculture supernatants or cell extracts containing EP2 or EP2 fragmentsare slowly passed through the column. The column is then washed withphosphate buffered saline together with appropriate membranesolubilizing such as detergents until the optical density (A₂₈₀) fallsto background, then the protein is eluted with 0.23M glycine-HCl (pH2.6) together with appropriate membrane solubilizing such as detergents.The purified EP2 protein is then dialyzed against phosphate bufferedsaline together with appropriate membrane solubilizing agent, such asdetergents.

One method suitable for the isolation of DNA encoding the prostaglandinreceptor of the present invention involves the utilization of amino acidand/or DNA sequence information obtained from other G-protein-linkedreceptors. Since other prostaglandin receptors are known to be G-proteinlinked, certain regions or domains such as the transmembrane and/orcytoplasmic domains, are expected to have some degree of homologysufficient to produce a probe for the isolation of novel receptors.

Prostaglandins and leukotrienes are known to transduce their signals viaG-protein-linked receptors. Distinct receptors for PGH₂ /thromboxane A₂,PGI₂, PGE₂, PGD₂, PGF2α, LTB₄, and LTD₄ present in various tissues havebeen described. Some of the receptors have been solubilized andpartially purified (Dutta-Roy, A. K. et al., (1987) JBC, 262, pp. 12685;Tsai, A. L. et al., (1989), JBC, 264, pp 61; 168--Watanabe, T. et. al.,(1990), JBC, 265, pp. 21237) and the human platelet TXA₂ receptor hasbeen purified to apparent homogeneity (Ushikubi, F. et. al., (1989),JBC, 264, pp. 16496). The purified thromboxane receptor exhibited a verybroad band on a SDS-polyacrylamide gel centered at appr. 57 kDa. Enoughprotein was obtained for partial sequence information.

An approach to the isolation of other eicosanoid receptor genes byhomology screening was taken, with the assumption that these receptorsare related in primary structure (Sugimoto, Y. et al., (1992), JBC, 267,pp. 6463). Since these receptors are of the G-protein-coupled receptorsuperfamily there are areas of homology which are likely to be found inthe transmembrane region and in the cytoplasmic domains. Therefore,various known G-protein linked receptors related to the prostaglandinreceptors may be utilized to provide DNA probes to regions of thereceptor protein-encoding DNA sought, which is likely to have homology,such as the transmembrane region.

Using a 0.68-kb fragment of a mouse EP2 receptor cDNA which encodes thec-terminal 165 amino acid region of this receptor was used to screen ahuman lung library from which a full-length human EP2 cDNA was isolated.This 1.958 kb cDNA clone encodes a 488-amino acid protein. This proteinwas designated as the EP2 receptor. Like many other G-protein coupledreceptors the EP2 receptor shares several common features. Firstly,there are 2 potential N-linked glycosylation sites at Asn7 and Asn177 inthe putative extracellular amino terminus. Secondly, conserved cysteineresidues are found in extracellular loops 1 and 2. There are multipleserine residues, potential sites of protein kinase phosphorylation,throughout the C-terminus and third cytoplasmic loops. The EP2 receptordoes not contain an aspartic acid residue in transmembrane three whichis characteristic of the receptors binding cationic amino-containingligands, however, it possesses a conserved arginine (position 315) foundin all known eicosanoid receptors within transmembrane seven. Thisregion is the most highly conserved among the eicosanoid receptors.

The novel prostaglandin receptor of the present invention is suitablefor use in an assay procedure for the identification of compounds whichmodulate the receptor activity. Modulating receptor activity, asdescribed herein includes the inhibition or activation of the receptorand also includes directly or indirectly affecting the normal regulationof the receptor activity. Compounds which modulate the receptor activityinclude agonists, antagonists and compounds which directly or indirectlyaffect regulation of the receptor activity.

The prostaglandin receptor of the present invention may be obtained fromboth native and recombinant sources for use in an assay procedure toidentify receptor modulators. In general, an assay procedure to identifyprostaglandin receptor modulators will contain the prostaglandinreceptor of the present invention, and a test compound or sample whichcontains a putative prostaglandin receptor modulator. The test compoundsor samples may be tested directly on, for example, purified receptorprotein whether native or recombinant, subcellular fractions ofreceptor-producing cells whether native or recombinant, and/or wholecells expressing the receptor whether native or recombinant. The testcompound or sample may be added to the receptor in the presence orabsence of a known labelled or unlabelled receptor ligand. Themodulating activity of the test compound or sample may be determined by,for example, analyzing the ability of the test compound or sample tobind to the receptor, activate the receptor, inhibit receptor activity,inhibit or enhance the binding of other compounds to the receptor,modify receptor regulation, or modify an intracellular activity.

The identification of modulators of EP2 receptor activity are useful intreating disease states involving the EP2 receptor activity. Othercompounds may be useful for stimulating or inhibiting activity of thereceptor. Selective agonists or antagonists of the EP2 receptor may beof use in the treatment of edema associated with inflammation, painresponse and fever, and may have utility as modulators of osteoclastfunction and hence bone resorbtion, T and B-lymphocyte function, andhence immunilogical reactions, smooth muscle relaxation, including thevarcular and trachiobronchial networks, and neoplastic and metastatictumor growth. The isolation and purification of an EP2-encoding DNAmolecule would be useful for establishing the tissue distribution of EP2receptors, studying changes in EP2 receptor expression in diseasestates, as well as establishing a process for identifying compoundswhich modulate EP2 receptor activity.

The following examples are provided for the purpose of illustrating thepresent invention without, however, limiting the same thereto.

EXAMPLE 1 Cloning of the EP2 cDNA

A mouse EP2 partial cDNA (680 bp) was obtained by RT-PCR from mousemastocytoma P-815 cell total RNA and cloned. This mouse EP2 fragment wasused to generate a ³² P-labeled cDNA probe to screen a human lung lambdagt10 library (Clontech, Palo Alto, Calif.) using standard techniques(Sambrook et al., 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

From this screening a 1.958 kb cDNA clone was plaque-purified and DNAwas prepared by the plate lysate method (Sambrook et al., 1989.Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

Subcloning and sequencing of cDNA

The 1.958 kb EcoRI fragment (EP2) was subcloned into pSK vector(Stratagene, La Jolla, Calif.) for sequencing using the T7 DNApolymerase sequencing kit (Pharmacia). The DNA was sequenced entirely onboth strands using the KS and SK primers (Stratagene, La Jolla, Calif.)or primers generated from the determined sequence. The nucleotidesequence of EP2 is shown in Table 1. The amino acid sequence for theencoded protein is shown in Table 2. The 1.958 kb fragment (EP2; FIG.1), when sequenced, was found to contain sequence homology to the humanEP1, EP3 and thromboxane receptor cDNA and the putative heptahelicalarrangement characteristic of G protein-coupled receptors was evident. Along open reading frame (1464 bp) was determined which would result in a488 amino acid polypeptide with a predicted relative molecular mass of53,115. The ATG assigned as the initiator codon matches the Kozakconsensus sequence for translation initiation (Kozak, 1989 J. Cell.Biol., 108, pp 229-241). There are 388 bp of 5'-untranslated sequenceincluding an in frame TGA stop codon 86 bp upstream of the predictedstart codon.

EXAMPLE 2 Construction of pcDNAIamp-EP2 expression vector

The 1.507 Kb Fsp1-Sca1 human EP2 cDNA fragment was subcloned into theEcoRV site of pcDNAIamp and the correct orientation was verified by PstI digestion.

                                      TABLE 1                                     __________________________________________________________________________    CGGCACAGCCTCACACCTGAACGCTGTCCTCCCGCAGACGAGACCGGCGGGCACTGCAAA                  GCTGGGACTCGTCTTTGAAGGAAAAAAAATAGCGAGTAAGAAATCCAGCACCATTCTTCA                  CTGACCCATCCCGCTGCACCTCTTGTTTCCCAAGTTTTTGAAAGCTGGCAACTCTGACCT                  CGGTGTCCAAAAATCGACAGCCACTGAGACCGGCTTTGAGAAGCCGAAGATTTGGCAGTT                  TCCAGACTGAGCAGGACAAGGTGAAAGCAGGTTGGAGGCGGGTCCAGGACATCTGAGGGC                  TGACCCTGGGGGCTCGTGAGGCTGCCACCGCTGCTGCCGCTACAGACCCAGCCTTGCACT                  CCAAGGCTGCGCACCGCCAGCCACTATCATGTCCACTCCCGGGGTCAATTCGTCCGCCTC                  CTTGAGCCCCGACCGGCTGAACAGCCCAGTGACCATCCCGGCGGTGATGTTCATCTTCGG                  GGTGGTGGGCAACCTGGTGGCCATCGTGGTGCTGTGCAAGTCGCGCAAGGAGCAGAAGGA                  GACGACCTTCTACACGCTGGTATGTGGGCTGGCTGTCACCGACCTGTTGGGCACTTTGTT                  GGTGAGCCCGGTGACCATCGCCACGTACATGAAGGGCCAATGGCCCGGGGGCCAGCCGCT                  GTGCGAGTACAGCACCTTCATTCTGCTCTTCTTCAGCCTGTCCGGCCTCAGCATCATCTG                  CGCCATGAGTGTCGAGCGCTACCTGGCCATCAACCATGCCTATTTCTACAGCCACTACGT                  GGACAAGCGATTGGCGGGCCTCACGCTCTTTGCAGTCTATGCGTCCAACGTGCTCTTTTG                  CGCGCTGCCCAACATGGGTCTCGGTAGCTCGCGGCTGCAGTACCCAGACACCTGGTGCTT                  CATCGACTGGACCACCAACGTGACGGCGCACGCCGCCTACTCCTACATGTACGCGGGCTT                  CAGCTCCTTCCTCATTCTCGCCACCGTCCTCTGCAACGTGCTTGTGTGCGGCGCGCTGCT                  CCGCATGCACCGCCAGTTCATGCGCCGCACCTCGCTGGGCACCGAGCAGCACCACGCGGC                  CGCGGCCGCCTCGGTTGCCTCCCGGGGCCACCCCGCTGCCTCCCCAGCCTTGCCGCGCCT                  CAGCGACTTTCGGCGCCGCCGGAGCTTCCGCCGCATCGCGGGCGCCGAGATCCAGATGGT                  CATCTTACTCATTGCCACCTCCCTGGTGGTGCTCATCTGCTCCATCCCGCTCGTGGTGCG                  AGTATTCGTCAACCAGTTATATCAGCCAAGTTTGGAGCGAGAAGTCAGTAAAAATCCAGA                  TTTGCAGGCCATCCGAATTGCTTCTGTGAACCCCATCCTAGACCCCTGGATATATATCCT                  CCTGAGAAAGACAGTGCTCAGTAAAGCAATAGAGAAGATCAAATGCCTCTTCTGCCGCAT                  TGGCGGGTCCCGCAGGGAGCGCTCCGGACAGCACTGCTCAGACAGTCAAAGGACATCTTC                  TGCCATGTCAGGCCACTCTCGCTCCTTCATCTCCCGGGAGCTGAAGGAGATCAGCAGTAC                  ATCTCAGACCCTCCTGCCAGACCTCTCACTGCCAGACCTCAGTGAAAATGGCCTTGGAGG                  CAGGAATTTGCTTCCAGGTGTGCCTGGCATGGGCCTGGCCCAGGAAGACACCACCTCACT                  GAGGACTTTGCGAATATCAGAGACCTCAGACTCTTCACAGGGTCAGGACTCAGAGAGTGT                  CTTACTGGTGGATGAGGCTGGTGGGAGCGGCAGGGCTGGGCCTGCCCCTAAGGGGAGCTC                  CCTGCAAGTCACATTTCCCAGTGAAACACTGAACTTATCAGAAAAATGTATATAATAGGC                  AAGGAAAGAAATACAGTACTGTTTCTGGACCCTTATAAAATCCTGTGCAATAGACACATA                  CATGTCACATTTAGCTGTGCTCAGAAGGGCTATCATCA  (SEQ. ID. NO.: 1)                     __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    MSTPGVNSSASLSPDRLNSPVTIPAVMFIFGVVGNLVAIVVLCKSRKEQKETTFYTLVCG                  LAVTDLLGTLLVSPVTIATYMKGQWPGGQPLCEYSTFILLFFSLSGLSIICAMSVERYLA                  INHAYFYSHYVDKRLAGLTLFAVYASNVLFCALPNMGLGSSRLQYPDTWCFIDWTTNVTA                  HAAYSYMYAGFSSFLILATVLCNVLVCGALLRMHRQFMRRTSLGTEQHHAAAAASVASRG                  HPAASPALPRLSDFRRRRSFRRIAGAEIQMVILLIATSLVVLICSIPLVVRVFVNQLYQP                  SLEREVSKNPDLQAIRIASVNPILDPWIYILLRKTVLSKAIEKIKCLFCRIGGSRRERSG                  QHCSDSQRTSSAMSGHSRSFISRELKEISSTSQTLLPDLSLPDLSENGLGGRNLLPGVPG                  MGLAQEDTTSLRTLRISETSDSSQGQDSESVLLVDEAGGSGRAGPAPKGSSLQVTFPSET                  LNLSEKCI  (SEQ. ID. NO.: 2)                                                   __________________________________________________________________________

EXAMPLE 3 Cloning of the EP2 cDNA into E. coli Expression Vectors

Recombinant EP2 is produced in E. coli following the transfer of the EP2expression cassette into E. coli expression vectors, including but notlimited to, the pET series (Novagen). The pET vectors place EP2expression under control of the tightly regulated bacteriophage T7promoter. Following transfer of this construct into an E. coli hostwhich contains a chromosomal copy of the T7 RNA polymerase gene drivenby the inducible lac promoter, expression of EP2 is induced when anappropriate lac substrate (IPTG) is added to the culture. The levels ofexpressed EP2 are determined by the assays described above.

The cDNA encoding the entire open reading frame for EP2 is inserted intothe NdeI site of pET 11a. Constructs in the positive orientation areidentified by sequence analysis and used to transform the expressionhost strain BL21. Transformants are then used to inoculate cultures forthe production of EP2 protein. Cultures may be grown in M9 or ZB media,whose formulation is known in the art. After growth to an approximateOD₆₀₀ =1.5, expression of EP2 is induced with 1 mM IPTG for 3 hours at37° C. EP2 receptor binding activity will be found in membrane fractionsfrom these cells.

EXAMPLE 4 In Vivo Translation of Synthetic EP2 mRNA by Xenopus OocyteMicroinjection and Expression in Mammalian Cells

EP2 cDNA constructs are ligated into in vitro transcription vectors (thepGEM series, Promega) for the production of synthetic mRNAs.

Synthetic mRNA is produced in sufficient quantity in vitro by cloningdouble stranded DNA encoding EP2 mRNA into a plasmid vector containing abacteriophage promoter, linearizing the plasmid vector containing thecloned EP2-encoding DNA, and transcribing the cloned DNA in vitro usinga DNA-dependent RNA polymerase from a bacteriophage that specificallyrecognizes the bacteriophage promoter on the plasmid vector.

Various plasmid vectors are available containing a bacteriophagepromoter recognized by a bacteriophage DNA-dependent RNA polymerase,including but not limited to plasmids pSP64, pSP65, pSP70, pSP71, pSP72,pSP73, pGEM-3Z, pGEM-4Z, pGEM-3Zf, pGEM-5Zf, pGEM-7Zf, pGEM-9Zf, andpGEM-11Zf, the entire series of plasmids is commercially available fromPromega.

The double stranded EP2-encoding DNA is cloned into the bacteriophagepromoter containing vector in the proper orientation using one or moreof the available restriction endonuclease cloning sites on the vectorwhich are convenient and appropriate for cloning EP2 DNA. The vectorwith the ligated EP2 DNA is used to transform bacteria, and clonalisolates are analyzed for the presence of the vector with the EP2 DNA inthe proper orientation.

Once a vector containing the EP2-encoding DNA in the proper orientationis identified and isolated, it is linearized by cleavage with arestriction endonuclease at a site downstream from, and withoutdisrupting, the EP2 transcription unit. The linearized plasmid isisolated and purified, and used as a template for in vitro transcriptionof EP2 mRNA.

The template DNA is then mixed with bacteriophage-specific DNA-dependentRNA polymerase in a reaction mixture which allows transcription of theDNA template forming EP2 mRNA. Several bacteriophage-specificDNA-dependent RNA polymerases are available, including but not limitedto T3, T7, and SP6 RNA polymerase. The synthetic EP2 mRNA is thenisolated and purified.

It may be advantageous to synthesize mRNA containing a 5' terminal capstructure and a 3' poly A tail to improve mRNA stability. A capstructure, or 7-methylguanosine, may be incorporated at the 5'terminusof the mRNA by simply adding 7-methylguanosine to the reaction mixturewith the DNA template. The DNA-dependent RNA polymerase incorporates thecap structure at the 5' terminus as it synthesizes the mRNA. The poly Atail is found naturally occurring in many cDNAs but can be added to the3' terminus of the mRNA by simply inserting a poly A tail-encoding DNAsequence at the 3' end of the DNA template.

The isolated and purified EP2 mRNA is translated using either acell-free system, including but not limited to rabbit reticulocytelysate and wheat germ extracts (both commercially available from Promegaand New England Nuclear) or in a cell based system, including but notlimited to microinjection into Xenopus oocytes, with microinjection intoXenopus oocytes being preferred.

Xenopus oocytes are microinjected with a sufficient amount of syntheticEP2 mRNA to produce EP2 protein. The microinjected oocytes are incubatedto allow translation of the EP2 mRNA, forming EP2 protein.

These synthetic mRNAs are injected into Xenopus oocytes (stage 5-6) bystandard procedures Gurdon, J. B. and Wickens, M. D. Methods in Enzymol.101:370-386, (1983)!. Oocytes are harvested and analyzed for EP2expression as described below.

EXAMPLE 5 pcDNAIamp-EP2 expression in Xenopus oocytes

Oocytes were taken from adult females of Xenopus laevis using standardsurgical procedure (Colman, A., 1984 In: Transcription andTranslation--A Practical Approach, IRL Press). To remove follicle cells,oocytes were treated for 2-3 h with freshly made collagenase (2 mg/ml,type 2, Worthington Biochemical Corp., Freehold, N.J.) in Ca²⁺ -freeND96 solution (ND96 in mM: NaCl 96, KCl 2, MgCl₂ 1, HEPES 5, Na-pyruvate2.5, theophylline 0.5 gentamicin 50 mg/ml, +1.8 CaCl₂, pH 7.6).Defolliculated stage 5-6 oocytes were selected and maintained in ND96solution. Oocyte nuclei were injected with 1.6 ng of pcDNAIamp-EP2 plus2.5 ng of pcDNAIamp-CFTR and then incubated at 18° C. for 48 h beforechallenge with agonist. CFTR (cystic fibrosis transmembrane regulator, acAMP dependent Cl⁻ channel) was co-expressed with EP2 receptor in theseoocytes and served as a reporter of changes in intracellular cAMPlevels. Functional activity was determined by measurement ofPGE2-induced CFTR-mediated Cl⁻ current. An oocyte was placed in a 0.5 mlperfusion chamber and voltage clamped at -60 mV (with microelectrodes of0.5-2.0 MW resistance filled with 3M KCl) using a Turbo TEC01C amplifier(NP1 Instruments, Germany). Ligand-containing solution was perfused andthe current response was recorded.

Perfusion of 1 nM PGE2 agonist, resulted in prominent current responsesin oocytes injected with pcDNAIamp-EP2 plus pcDNAIamp-CFTR confirmingthat this clone encodes a functional EP2 receptor that is coupled to thecAMP signalling pathway (FIG. 3). The response to 1 μM PGF₂α was muchsmaller as expected for the EP2 receptor subtype. Such responses wereabsent in control (CFTR alone or CFTR plus antisense EP2 cDNA injected)oocytes (FIG. 4). This rank order of potency is consistent with thatreported for the EP2 receptor Coleman, et al., 1991!.

EXAMPLE 6 Cloning of EP2 cDNA into a Mammalian Expression Vector

EP2 cDNA expression cassettes are ligated at appropriate restrictionendonuclease sites to the following vectors containing strong, universalmammalian promoters: pBC12BI Cullen, B. R. Methods in Enzymol. 152:684-704 1988!, and pEE12 (CellTech EP O 338,841) and its derivativespSZ9016-1 and p9019. p9019 represents the construction of a mammalianexpression vector containing the hCMVIE promoter, polylinker and SV40polyA element with a selectable marker/amplification system comprised ofa mutant gene for dihydrofolate reductase (mDHFR) (Simonsen, C. C. andLevinson, A. D. Proc. Natl. Acad. Sci USA 80: 2495-2499 1983!) driven bythe SV40 early promoter. An SV40 polyadenylation sequence is generatedby a PCR reaction defined by primers 13978-120 and 139778-121 using pD5(Berker and Sharp, Nucl. Acid Res. 13: 841-857 1985!) as template. Theresulting 0.25 Kb PCR product is digested with ClaI and SpeI and ligatedinto the 6.7 Kb fragment of pEE 12 which had been likewise digested. Theresultant plasmid is digested with BglII and SfiI to liberate the 3'portion of the SV40 early promoter and the GScDNA from the vector. A0.73 Kb SfiI-XhoII fragment isolated from plasmid pFR400 (Simonsen, C.C. and Levinson, A. D. Proc. Natl. Acad. Sci USA 80: 2495-2499 1983!) isligated to the 5.6 Kb vector described above, reconstituting the SV40early promoter, and inserting the mdHFR gene. This plasmid is designatedp9019. pSZ9016-1 is identical to p9019 except for the substitution ofthe HIV LTR for the huCMVIE promoter. This vector is constructed bydigesting p9019 with XbaI and MluI to remove the huCMVIE promoter. TheHIV LTR promoter, from residue -117 to +80 (as found in the vector pCD23containing the portion of the HIV-1 LTR (Cullen, Cell 46:973 1986!) isPCR amplified from the plasmid pCD23 using oligonucleotide primers whichappended to the ends of the product the MluI and SpeI restriction siteson the 5' side while Hind III and Xba I sites are appended on the 3'side. Following the digestion of the resulting 0.2 kb PCR product withthe enzymes MluI and Xba I the fragment is agarose gel-purified andligated into the 4.3 Kb promoterless DNA fragment to generate the vectorpSZ9016- 1.

Cassettes containing the EP2 cDNA in the positive orientation withrespect to the promoter are ligated into appropriate restriction sites3' of the promoter and identified by restriction site mapping and/orsequencing. These cDNA expression vectors are introduced into varioushost cells including, but not limited to: COS-7 (ATCC# CRL1651), CV-1Sackevitz et al., Science 238: 1575 (1987)!, 293, L cells (ATCC#CRL6362)! by standard methods including but not limited toelectroporation, or chemical procedures (cationic liposomes, DEAEdextran, calcium phosphate). Transfected cells and cell culture extractscan be harvested and analyzed for EP2 expression as described below.

All of the vectors used for mammalian transient expression can be usedto establish stable cell lines expressing EP2. Unaltered EP2 cDNAconstructs cloned into expression vectors will be expected to programhost cells to make intracellular EP2 protein. The transfection hostcells include, but are not limited to, CV-1 Sackevitz et al., Science238: 1575 (1987)!, tk-L Wigler, et al. Cell 11: 223 (1977)!, NS/0, anddHFr-CHO Kaufman and Sharp, J. Mol. Biol. 159: 601, (1982)!.

Co-transfection of any vector containing EP2 cDNA with a drug selectionplasmid including, but not limited to G418, aminoglycosidephosphotransferase, pLNCX Miller, A. D. and Rosman G. J. Biotech News 7:980-990 (1989)!; hygromycin, hygromycin-B phosphotransferase, pLG90Gritz. L. and Davies, J., GENE 25: 179 (1983)!; APRT, xanthine-guaninephosphoribosyl-transferase, pMAM (Clontech) Murray, et al., Gene 31: 233(1984)! will allow for the selection of stably transfected clones.Levels of EP2 are quantitated by the assays described above.

EP2 cDNA constructs are ligated into vectors containing amplifiabledrug-resistance markers for the production of mammalian cell clonessynthesizing the highest possible levels of EP2. Following introductionof these constructs into cells, clones containing the plasmid areselected with the appropriate agent, and isolation of an over-expressingclone with a high copy number of the plasmid is accomplished byselection in increasing doses of the agent. The following systems areutilized: the 9016 or the 9019 plasmid containing the mutant DHFR geneSimonson, C. and Levinson, A., Proc. Natl. Acad. Sci. USA 80: 2495(1983)!, transfected into DHFR-CHO cells and selected in methotrexate;the pEE12 plasmid containing the glutamine synthetase gene, transfectedinto NS/O cells and selected in methionine sulfoximine (CellTechInternational Patent Application 2089/10404); and 9016 or other CMVpromoter vectors, co-transfected with pDLAT-3 containing the thymidinekinase gene Colbere and Garopin, F., Proc. Natl. Acad. Sci. 76: 3755(1979)! in APRT and TK deficient L cells, selected in APRT (0.05 mMazaserine, 0.1 mM adenine, 4 ug/ml adenosine) and amplified with HAT(100 uM hypoxanthine, 0.4 uM aminopterin, 16 uM thymidine).

EXAMPLE 7 Expression of the EP2 receptor in COS-M6 cells and ³ H!PGE2binding assays

The recently cloned human prostaglandin E2 (EP2) receptor was subclonedinto the pcDNAlamp plasmid (Invitrogen) and transfected into COS-M6cells using the DEAE-dextran method. The cells were maintained inculture for 72 h, then harvested and membranes prepared by differentialcentrifugation (1000×g for 10 min, then 100,000×g for 30 min) followinglysis of the cells by nitrogen cavitation. ³ H!Prostaglandin E2 ( ³H!PGE2) binding assays were performed in 10 mM MES/KOH pH 6.0,containing 1.0 mM EDTA, 10 mM MnCl₂, 0.3 nM ³ H!PGE2 and 12-15 μg ofprotein from the 100,000×g membrane fraction. Incubations were conductedfor 45 min at 30° C. prior to separation of the bound and freeradioligand by rapid filtration through Whatman GF/B filters presoakedat 4° C. in washing buffer (10 μM MES/KOH (pH 6.0) containing 0.01%bovine serum albumin). The filters were washed with approximately 16 mlof washing buffer and the residual ³ H!PGE2 bound to the filter wasquantified by liquid scintillation counting. Specific binding wasdefined as the difference between total binding and non-specificbinding, determined in the presence of 2 μM PGE2.

The cloned human EP2 receptor was transfected into COS-M6 cells and ³H!PGE2 binding assays were performed with membranes prepared from thetransfected cells. In competition assays PGE2 and PGE1 were the mostpotent competing ligands with IC50 values of 1 nM (FIG. 5). The rankorder of potency for prostaglandins and related analogs was:PGE2=PGE1>>phenyl-trinor PGE2>iloprost>PGF₂α >PGD₂ ≈U46619. U46619 andiloprost are stable analogs of thromboxane and prostacyclin and displaycomparable potency at the TP and IP receptors, respectively. Inaddition, the EP3 agonist MB28767 was approximately 30-fold less potentat EP2 than EP3, the EP1 antagonist AH6809 and SC19220 were essentiallyinactive at EP2 and butaprost, an EP2 agonist was also relativelyinactive with an IC₅₀ of 30 μM. Misoprostol, a gastrointestinalprotective agent had an IC₅₀ of 6.03 μM. This rank order of potency hasbeen predicted for the EP2 receptor from previous pharmacologicalstudies.

EXAMPLE 8 Cloning of EP2 cDNA into a Baculovirus Expression Vector forExpression in Insect Cells

Baculovirus vectors, which are derived from the genome of the AcNPVvirus, are designed to provide high level expression of cDNA in the Sf9line of insect cells (ATCC CRL# 1711). Recombinant baculovirusesexpressing EP2 cDNA are produced by the following standard methods (InVitrogen Maxbac Manual): the EP2 cDNA constructs are ligated downstreamof the polyhedrin promoter in a variety of baculovirus transfer vectors,including the pAC360 and the pBlueBac vector (In Vitrogen). Recombinantbaculoviruses are generated by homologous recombination followingco-transfection of the baculovirus transfer vector and linearized AcNPVgenomic DNA Kitts, P. A., Nuc. Acid. Res. 18: 5667 (1990)! into Sf9cells. Recombinant pAC360 viruses are identified by the absence ofinclusion bodies in infected cells (Summers, M. D. and Smith, G. E.,Texas Agriculture Exp. Station Bulletin No. 1555) and recombinantpBlueBac viruses are identified on the basis of β-galactosidaseexpression (Vialard, et al. 1990, J. Virol., 64, pp 37-50). Followingplaque purification and infection of sf9 cells with EP2 recombinantbaculovirus, EP2 expression is measured by the assays described above.

The cDNA encoding the entire open reading frame for EP2 is inserted intothe BamHI site of pBlueBacII. Constructs in the positive orientationwith respect to the polyhedrin promoter are identified by sequenceanalysis and used to transfect Sf9 cells in the presence of linear AcNPVwild type DNA.

Authentic, active EP2 is found associated with the membranes of infectedcells. Membrane preparations are prepared from infected cells bystandard procedures.

EXAMPLE 9 Cloning of EP2 cDNA into a yeast expression vector

Recombinant EP2 is produced in the yeast S. cerevisiae following theinsertion of the optimal EP2 cDNA construct into expression vectorsdesigned to direct the intracellular expression of heterologousproteins. For intracellular expression, vectors such as EmBLyex4 or thelike are ligated to the EP2 cistron Rinas, U. et al., Biotechnology 8:543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265: 4189-4192(1989)!. The levels of expressed EP2 are determined by the assaysdescribed above.

EXAMPLE 10 Purification of Recombinant EP2

Recombinantly produced EP2 may be purified by antibody affinitychromatography.

EP2 antibody affinity columns are made by adding the anti-EP2 antibodiesto Affigel-10 (Biorad), a gel support which is pre-activated withN-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) together withappropriate membrane solubilizing agents such as detergents and the cellculture supernatants or cell extracts containing solubilized EP2 isslowly passed through the column. The column is then washed withphosphate-buffered saline together with detergents until the opticaldensity (A280) falls to background, then the protein is eluted with0.23M glycine-HCl (pH 2.6) together with detergents. The purified EP2protein is then dialyzed against phosphate buffered saline together withdetergents.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1958 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGGCACAGCCTCACACCTGAACGCTGTCCTCCCGCAGACGAGACCGGCGGGCACTGCAAA60                GCTGGGACTCGTCTTTGAAGGAAAAAAAATAGCGAGTAAGAAATCCAGCACCATTCTTCA120               CTGACCCATCCCGCTGCACCTCTTGTTTCCCAAGTTTTTGAAAGCTGGCAACTCTGACCT180               CGGTGTCCAAAAATCGACAGCCACTGAGACCGGCTTTGAGAAGCCGAAGATTTGGCAGTT240               TCCAGACTGAGCAGGACAAGGTGAAAGCAGGTTGGAGGCGGGTCCAGGACATCTGAGGGC300               TGACCCTGGGGGCTCGTGAGGCTGCCACCGCTGCTGCCGCTACAGACCCAGCCTTGCACT360               CCAAGGCTGCGCACCGCCAGCCACTATCATGTCCACTCCCGGGGTCAATTCGTCCGCCTC420               CTTGAGCCCCGACCGGCTGAACAGCCCAGTGACCATCCCGGCGGTGATGTTCATCTTCGG480               GGTGGTGGGCAACCTGGTGGCCATCGTGGTGCTGTGCAAGTCGCGCAAGGAGCAGAAGGA540               GACGACCTTCTACACGCTGGTATGTGGGCTGGCTGTCACCGACCTGTTGGGCACTTTGTT600               GGTGAGCCCGGTGACCATCGCCACGTACATGAAGGGCCAATGGCCCGGGGGCCAGCCGCT660               GTGCGAGTACAGCACCTTCATTCTGCTCTTCTTCAGCCTGTCCGGCCTCAGCATCATCTG720               CGCCATGAGTGTCGAGCGCTACCTGGCCATCAACCATGCCTATTTCTACAGCCACTACGT780               GGACAAGCGATTGGCGGGCCTCACGCTCTTTGCAGTCTATGCGTCCAACGTGCTCTTTTG840               CGCGCTGCCCAACATGGGTCTCGGTAGCTCGCGGCTGCAGTACCCAGACACCTGGTGCTT900               CATCGACTGGACCACCAACGTGACGGCGCACGCCGCCTACTCCTACATGTACGCGGGCTT960               CAGCTCCTTCCTCATTCTCGCCACCGTCCTCTGCAACGTGCTTGTGTGCGGCGCGCTGCT1020              CCGCATGCACCGCCAGTTCATGCGCCGCACCTCGCTGGGCACCGAGCAGCACCACGCGGC1080              CGCGGCCGCCTCGGTTGCCTCCCGGGGCCACCCCGCTGCCTCCCCAGCCTTGCCGCGCCT1140              CAGCGACTTTCGGCGCCGCCGGAGCTTCCGCCGCATCGCGGGCGCCGAGATCCAGATGGT1200              CATCTTACTCATTGCCACCTCCCTGGTGGTGCTCATCTGCTCCATCCCGCTCGTGGTGCG1260              AGTATTCGTCAACCAGTTATATCAGCCAAGTTTGGAGCGAGAAGTCAGTAAAAATCCAGA1320              TTTGCAGGCCATCCGAATTGCTTCTGTGAACCCCATCCTAGACCCCTGGATATATATCCT1380              CCTGAGAAAGACAGTGCTCAGTAAAGCAATAGAGAAGATCAAATGCCTCTTCTGCCGCAT1440              TGGCGGGTCCCGCAGGGAGCGCTCCGGACAGCACTGCTCAGACAGTCAAAGGACATCTTC1500              TGCCATGTCAGGCCACTCTCGCTCCTTCATCTCCCGGGAGCTGAAGGAGATCAGCAGTAC1560              ATCTCAGACCCTCCTGCCAGACCTCTCACTGCCAGACCTCAGTGAAAATGGCCTTGGAGG1620              CAGGAATTTGCTTCCAGGTGTGCCTGGCATGGGCCTGGCCCAGGAAGACACCACCTCACT1680              GAGGACTTTGCGAATATCAGAGACCTCAGACTCTTCACAGGGTCAGGACTCAGAGAGTGT1740              CTTACTGGTGGATGAGGCTGGTGGGAGCGGCAGGGCTGGGCCTGCCCCTAAGGGGAGCTC1800              CCTGCAAGTCACATTTCCCAGTGAAACACTGAACTTATCAGAAAAATGTATATAATAGGC1860              AAGGAAAGAAATACAGTACTGTTTCTGGACCCTTATAAAATCCTGTGCAATAGACACATA1920              CATGTCACATTTAGCTGTGCTCAGAAGGGCTATCATCA1958                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 488 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerThrProGlyValAsnSerSerAlaSerLeuSerProAspArg                              151015                                                                        LeuAsnSerProValThrIleProAlaValMetPheIlePheGlyVal                              202530                                                                        ValGlyAsnLeuValAlaIleValValLeuCysLysSerArgLysGlu                              354045                                                                        GlnLysGluThrThrPheTyrThrLeuValCysGlyLeuAlaValThr                              505560                                                                        AspLeuLeuGlyThrLeuLeuValSerProValThrIleAlaThrTyr                              65707580                                                                      MetLysGlyGlnTrpProGlyGlyGlnProLeuCysGluTyrSerThr                              859095                                                                        PheIleLeuLeuPhePheSerLeuSerGlyLeuSerIleIleCysAla                              100105110                                                                     MetSerValGluArgTyrLeuAlaIleAsnHisAlaTyrPheTyrSer                              115120125                                                                     HisTyrValAspLysArgLeuAlaGlyLeuThrLeuPheAlaValTyr                              130135140                                                                     AlaSerAsnValLeuPheCysAlaLeuProAsnMetGlyLeuGlySer                              145150155160                                                                  SerArgLeuGlnTyrProAspThrTrpCysPheIleAspTrpThrThr                              165170175                                                                     AsnValThrAlaHisAlaAlaTyrSerTyrMetTyrAlaGlyPheSer                              180185190                                                                     SerPheLeuIleLeuAlaThrValLeuCysAsnValLeuValCysGly                              195200205                                                                     AlaLeuLeuArgMetHisArgGlnPheMetArgArgThrSerLeuGly                              210215220                                                                     ThrGluGlnHisHisAlaAlaAlaAlaAlaSerValAlaSerArgGly                              225230235240                                                                  HisProAlaAlaSerProAlaLeuProArgLeuSerAspPheArgArg                              245250255                                                                     ArgArgSerPheArgArgIleAlaGlyAlaGluIleGlnMetValIle                              260265270                                                                     LeuLeuIleAlaThrSerLeuValValLeuIleCysSerIleProLeu                              275280285                                                                     ValValArgValPheValAsnGlnLeuTyrGlnProSerLeuGluArg                              290295300                                                                     GluValSerLysAsnProAspLeuGlnAlaIleArgIleAlaSerVal                              305310315320                                                                  AsnProIleLeuAspProTrpIleTyrIleLeuLeuArgLysThrVal                              325330335                                                                     LeuSerLysAlaIleGluLysIleLysCysLeuPheCysArgIleGly                              340345350                                                                     GlySerArgArgGluArgSerGlyGlnHisCysSerAspSerGlnArg                              355360365                                                                     ThrSerSerAlaMetSerGlyHisSerArgSerPheIleSerArgGlu                              370375380                                                                     LeuLysGluIleSerSerThrSerGlnThrLeuLeuProAspLeuSer                              385390395400                                                                  LeuProAspLeuSerGluAsnGlyLeuGlyGlyArgAsnLeuLeuPro                              405410415                                                                     GlyValProGlyMetGlyLeuAlaGlnGluAspThrThrSerLeuArg                              420425430                                                                     ThrLeuArgIleSerGluThrSerAspSerSerGlnGlyGlnAspSer                              435440445                                                                     GluSerValLeuLeuValAspGluAlaGlyGlySerGlyArgAlaGly                              450455460                                                                     ProAlaProLysGlySerSerLeuGlnValThrPheProSerGluThr                              465470475480                                                                  LeuAsnLeuSerGluLysCysIle                                                      485                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated and purified prostaglandin receptorprotein wherein said protein comprises the amino acid sequence ##STR1##2. A method of determining whether a test compound binds to aprostaglandin receptor, comprising:(a) providing, mammalian host cellstransfected with a nucleic acid encoding a prostaglandin receptorwherein said receptor comprises the amino acid sequence of claim 1; (b)cultivating said mammalian host cells under conditions such that saidreceptor is expressed, (c) combining said test compound with saidmammalian host cells or with membranes containing said prostaglandinreceptor prepared from said mammalian host cells; and (d) determiningwhether said test compound binds to said prostaglandin receptor.
 3. Amethod of determining whether a test compound is an agonist of aprostaglandin receptor, comprising:(a) introducing a nucleic acidencoding a prostaglandin receptor comprising the amino acid sequence ofclaim 1 and a nucleic acid encoding the cystic fibrosis transmembraneregulator into Xenopus oocytes; (b) cultivating said oocytes underconditions such that the prostaglandin receptor and the cystic fibrosistransmembrane regulator are expressed; (c) combining the test compoundwith the oocytes of step (b); (d) measuring the Cl⁻ current in theoocytes following combination with the test compound; where an increasein the Cl⁻ current in the oocytes following combination with the testcompound indicates that the test compound is an agonist of theprostaglandin receptor.